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CARDIOVASCULAR ANESTHESIA

The Plasma Supplemented Modified Activated Clotting Time for Monitoring of Heparinization During Cardiopulmonary Bypass: A Pilot Investigation

Andreas Koster, MD^*, George Despotis, MD, Marcus Gruendel, MD^*, Thomas Fischer, MD^*, Michael Praus, MD, Herman Kuppe, MD^*, and Jerrold H. Levy, MD

*Department of Anesthesia, Deutsches Herzzentrum, and Institute of Pathobiochemistry and Clinical Chemistry, Charité, Campus Virchow, Berlin, Germany; Departments of Anesthesiology, Pathology, and Immunology, Washington University School of Medicine, St Louis, Missouri; and Department of Cardiothoracic Anesthesiology, Emory University School of Medicine, Atlanta, Georgia

Address correspondence and reprint requests to Andreas Koster, MD, Deutsches Herzzentrum Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. Address e-mail to Koster{at}dhzb.de

	Abstract

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The standard celite or kaolin activated clotting time (ACT) correlates poorly with heparin levels during cardiopulmonary bypass (CPB). We compared a modified kaolin ACT, in which plasma was supplemented, to a standard undiluted kaolin ACT for monitoring heparin levels during CPB. Fifteen patients undergoing normothermic CPB were enrolled in this prospective study. Heparin management was performed according to the Hepcon HMS results (Medtronic, Minneapolis, MN). The ACTs were performed with the ACT II device (Medtronic). Hepcon HMS calculations, standard kaolin ACTs, and plasma supplemented modified ACTs (mACTs), prepared by diluting blood samples 1:1 with human plasma (Behring, Marburg, Germany), were measured every 30 min during CPB. The data obtained were correlated to the plasma chromogenic anti-Xa activity as a reference assay for heparin levels. A total of 64 samples were evaluated. The chromogenic anti-Xa activity ranged from 0.2 to 5.5 IU/mL. The Hepcon HMS calculations ranged from 2.7–8.2 IU/mL of heparin, the standard ACT ranged from 424 to >999 s, and the mACT ranged from 210 to 801 s. The correlation to the chromogenic anti-Xa method was r = 0.43 for the standard kaolin ACT and r = 0.69 for the plasma mACT. The plasma mACT provided an improved correlation to chromogenically measured levels of anti-Xa activity during CPB. The improved correlation most likely results from a correction of the effects of the impairment of the coagulation system caused by hemodilution and consumption of procoagulants on extracorporeal surfaces.

IMPLICATIONS: During cardiopulmonary bypass, the plasma modified kaolin activated clotting time (ACT) provides a better correlation with heparin levels than the standard kaolin ACT.

	Introduction

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Hemostatic activation during cardiac surgery can occur through multiple mechanisms. The contact of blood with nonendothelial surfaces of cardiopulmonary bypass (CPB) concurrent with the massive release and reinfusion (e.g., pericardial suction) of tissue factor from surgical trauma, despite systemic anticoagulation, leads to the activation of the coagulation system. As a result, thrombin is generated, plasma coagulation factors and platelets are consumed, and fibrinolysis and inflammatory responses are initiated. The postperfusion syndrome and hemostatic defect of CPB are clinical consequences of these processes (1,2).

Several clinical investigations have suggested that the maintenance of large heparin concentrations during CPB results in an improved preservation of the coagulation system, reduced blood loss, and transfusion requirements (3–6). The standard assay for monitoring anticoagulation during cardiac surgery is the activated clotting time (ACT). The activators currently used for the ACT, kaolin and celite, initiate contact activation. Therefore, ACT is more indicative of inhibition of contact activation rather than providing information about the degree of inhibition of the extrinsic coagulation pathway and heparin levels during CPB (7–9). Further, with prolonged hemostatic activation during CPB, the ACT can be prolonged, although heparin levels decrease, which might result in the potential for further hemostatic activation and progressive consumptive coagulopathy.

The only currently available point-of-care device for monitoring heparin levels, the Hepcon HMS (Medtronic, Minneapolis, MN), measures heparin concentrations in multi-channel cartridges via an automated protamine titration method using a thromboplastin reagent. However, there is some controversy with regard to the precision of the results obtained (10,11). A modified Heptest (Haemachem, Inc, St Louis, MO) for monitoring heparin anti-Xa activity in whole blood was introduced and provided a good correlation to the heparin levels during CPB (12). Nevertheless, this new method requires validation in larger clinical trials, and a ready-to-use test is not commercially available.

Investigations have shown that high (approximately 50%–70%) levels increase the reliability of monitoring other anticoagulants such as hirudin (13). The supplementation of plasma or prothrombin to the ecarin clotting time is an established procedure to improve the reliability of this assay to monitor direct thrombin inhibitors, such as hirudin, during extracorporeal circulation (14). Moreover, it was demonstrated that a plasma modified ACT (mACT) provided a good correlation to r-hirudin levels measured during the condition of CPB (15).

Based on these considerations, we investigated whether we could improve the ability to monitor unfractionated heparin (UFH) during CPB using a mACT, providing a minimum of 50% coagulation factors by adding 1:1 volumes of plasma to the sample.

	Materials and Methods

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After approval by the local ethics committee and informed consent, 15 patients undergoing coronary artery bypass grafting were studied in this prospective investigation. After IV access and placement of an arterial catheter, anesthesia was induced with etomidate, midazolam, sufentanil, and pancuronium bromide. Anesthesia was maintained using a total IV technique with a continuous infusion of propofol and sufentanil supplemented by pancuronium. Additionally, vasoactive drugs and inotropic drugs were administered by demand. Normothermic CPB was performed with nonheparin-coated systems and the use of membrane oxygenators. In none of the patients were aprotinin or other antifibrinolytic drugs administered.

Anticoagulation was performed, following the institutional standard, according to the results of the Hepcon HMS. A kaolin ACT (kACT) of 480 s was determined as the target value of the heparin-dose-response (HDR) cartridge for individual calculation of the heparin level required to achieve this ACT. The HDR was performed before skin incision. After the administration of the heparin, an ACT (Hepcon HMS kACT) was obtained before the initiation of CPB. During CPB, the target heparin levels were obtained at intervals of 30 min and maintained according to the results of the six-channel heparin-protamine-titration (HPT) cartridge. If results were at the test cartridge’s upper or lower limits, measurements were repeated with higher range or lower range cartridges if possible. After the conclusion of CPB, the protamine dosage required to reverse patient and CPB heparin was calculated according to the results of the HPT measurement. After the protamine administration and infusion of the total volume of the CPB circuit, residual heparin was measured using the low range (0–1.5 IU/mL of heparin) cartridge.

During CPB, kACT measurements using the ACT II device (Medtronic) and plasma mACTs were performed in parallel with the HPT measurements. For both assays, the mean value of the duplicate measurement was used for further statistical calculations. If the difference between the two channels was more than 10%, all measurements were repeated. For measurement of the mACT, 1 mL of patient blood was mixed with 1 mL of standard human plasma (Behring, Marburg, Germany) and then transferred into the ACT II cartridges. Because of limitations of the ACT II device, measurements were automatically interrupted after 999 s.

Additionally, in parallel to every ACT measurement during CPB, 5 mL of citrated whole blood was obtained for the determination of the plasma anti-Xa activity using a chromogenic assay. The chromogenic assay was performed on an STA analyzer (Roche Diagnostics, Mannheim, Germany) that used the STA LMWH test kit (Roche Diagnostics). Calibration curves were performed with calibrator plasma (Hepanorm^® H, Diagnostica Stago, Asneirs-sur-Seine, France). Fifty microliters of the sample were diluted with 50 µL of a diluent buffer, and 100 µL of Factor Xa and 100 µL of substrate were added. For kinetic measurement of the remaining Factor Xa, the change in optical density was assessed at 405 nm. Quality control of the test was performed in a preliminary investigation. Linearity was achieved to 6.0 IU/mL of anti-Xa activity. The intra-assay variability revealed a variation coefficient of 3.5%.

If measurements with the ACT II device exceeded 999 s, the value of 999 s was used for statistical analysis. The Gaussian normal distribution of the obtained values was assessed using the Kolmogorov-Smirnov test (Statistical Package for Social Science, version 10.0.5, SPSS, Chicago, IL). Normal distribution of the values obtained was proved for the variables of the anti-Xa test for mACT but not for the ACT. The correlation of the ACT and mACT to the non-hematocrit-(Hct) corrected chromogenic reference anti-Xa assay was evaluated using Spearman’s rank order correlation. Agreement (disagreement) of the values of the ACT and mACT was evaluated using the method of Bland and Altman.

	Results

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There were nine male and six female patients. Their age ranged from 35–82 yr and body weight from 47–105 kg. Among the 15 patients, 7 patients received preoperative IV heparin (activated partial thromboplastin time, 40–60 s) and 3 patients received aspirin (1 x 100 mg/d) until surgery. In the remaining 5 patients, a subcutaneous bolus injection of 7500 IU of UFH twice a day was given until surgery.

The duration of CPB ranged from 63 to 185 min. A total of 64 samples were obtained, and 256 measurements were performed. Repeat measurements were required in three cases because the difference between the duplicate measurements of the kACT exceeded 10%. Systemic anticoagulation required heparin target levels between 2.7 and 7.5 IU/mL according to the HDR calculation. The dosages required to achieve these values ranged from 17.000 to 67.000 IU (3.4 to 8.2 IU/kg) with a mean ± SD of 34.000 ± 12.000. The total heparin requirement ranged from 29.000 to 97.000 IU with a mean of 54.000 ± 17.000 IU (4.1 to 13.5 IU/kg).

The Hct values ranged from 19% to 32% with a mean of 27 ± 4, and the platelet counts ranged from 45,000 to 298,000/µL with a mean of 163,000 ± 31,000/µL. The results of the chromogenic anti-Xa assay ranged from 0.2 to 5.5 IU/mL, and the results of the Hepcon HMS ranged from 2.7 to 8.2 IU/mL. The kACT ranged from 424 to 999 s, and the mACT ranged from 210 to 725 s. In 14 of 64 samples, the kACT was prolonged for 999 s. The correlation to the non-Hct-corrected values of the chromogenic assay was r = 0.43 for the kACT (Fig. 1) (0.5 if values exceeding 999 s were excluded) and r = 0.69 for the mACT (Fig. 2). The Bland and Altman plot of mean versus difference of the ACT and mACT revealed a mean of 270.19 ± 110.70 ( Fig. 3).

View larger version (16K): [in this window] [in a new window]   Figure 1. Correlation of the kaolin activated clotting time (kACT) to the chromogenically measured plasma anti-Xa activity.

View larger version (16K): [in this window] [in a new window]   Figure 2. Correlation plasma added modified activated clotting time (mACT) to the chromogenically measured plasma anti-Xa activity.

View larger version (16K): [in this window] [in a new window]   Figure 3. Evaluation of agreement between the activated clotting time (ACT) and plasma added modified ACT (mACT) with the Bland and Altman test of mean versus difference.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Over the last few years, efforts have been made to improve the ACT to better correlate with heparin concentrations during CPB. Kaolin, a more potent stimulator of contact activation, was introduced as an alternative to celite as an activator. The MAX-ACT is a new ACT that uses maximal stimulation of factor XII. Clinical investigations revealed that this new ACT, under the conditions of CPB, was less influenced by temperature and Hct, but the lack of a close correlation to the heparin concentrations remained a limitation of this assay (16).

The supplementation of plasma coagulation factors to the kACT resulted in a completely different test result, as observed in the disagreement between both assays shown in the Bland and Altman calculation (Fig. 3), with improvement correlation of the mACT to heparin anti-Xa activity ranging from r = 0.43 to r = 0.69. Notably, all measurements of the mACT were within the range (<1000 seconds) of the device, whereas approximately 20% of the ACTs exceeded 999 seconds and therefore gave no valid value.

These data confirm that, comparable to the ecarin clotting time for monitoring direct thrombin inhibitors, the depletion of the plasma coagulation system by hemodilution and consumption has a major influence on the CPB defect of the ACT. It further demonstrates that significant improvement can be easily achieved by the supplementation of procoagulants to the assay.

The values of the plasma chromogenic assay were not corrected to the Hct values of the whole blood to avoid further imprecision by additional measurement of the Hct value, which would have been required for such correction. Therefore, the values of the plasma chromogenic assay, in contrast to the constant volume of the whole blood, are influenced by the Hct because it determines the volume in which the UFH are dissolved. Apart from the individual response to heparin that is caused, for example, by different levels of antithrombin, cell binding, or binding to platelet factor IV in the sample, these variations of the Hct (19%–32%) contribute to the scatter along the correlation curves in Figures 1 and 2.

However, it can be questioned whether maintenance of patient-specific heparin levels (used as a surrogate for effective anticoagulation) should be the primary end point for analysis of a new test system. Using the Hepcon HMS HDR cartridge, the heparin level necessary to achieve sufficient inhibition of contact activation is calculated in the condition of a non-affected coagulation system before surgery and CPB. With decreasing antithrombin levels during CPB and a possibly impaired heparin response (which is not detected by the protamine titration method), maintaining a specific heparin level may not provide sufficient anticoagulation. Therefore, a cartridge for this system that provides further information about the antithrombin activity is highly desirable. The mACT seems to offer great potential in this regard because it enables both adequate assessment and consecutive maintenance of heparin levels and, as a functional assay, the ability to monitor the inhibition of coagulation.

Although the mACT in its current form is easy and quick to perform, the need for precise operating steps during surgery limits its value as a point-of-care assay. Before routine use in clinical practice, further automation of the assay is highly desirable. However, this may be easily achieved by automated dispersion of the plasma replete to the sample by the addition of lyophilized plasma into the reagent chamber or by using specimen collection tubes that contain lyophilized plasma.

Limitations of the investigation are the limited number of data obtained and the use of a correlation between two assays as the end point of the study. For these reasons, it has been deemed a pilot investigation. Moreover, future investigations have to evaluate the target value for this test, which should then indicate improved inhibition of activation of the coagulation system during CPB. Thereafter, further clinical investigations will be required to demonstrate whether monitoring of anticoagulation during CPB according to the results obtained by the mACT significantly decreases the detrimental effect of CPB on the coagulation and inflammatory system and contributes to an improved patient outcome.

	Acknowledgments

 
Supported by the Deutsches Herzzentrum, Berlin, Germany.

	Footnotes

 
Previously presented at the IARS meeting, Fort Lauderdale, Florida, March 19, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999